Restriction Digests

There are two parts to this restriction digest lab:

  • A virtual digest, in which you use a computer and some paper to analyze DNA sequences and predict the results of restriction digests. Do this exercise in lab with your group and turn it in today, before you do the actual restriction digest.
  • A real restriction digest, in which you cut your purified plasmid DNA with restriction enzymes. In the next lab period, you'll analyze your digested DNA using electrophoresis.

In this lab you'll use restriction enzymes to cut your two purified plasmids at specific sites. You'll also cut some λ DNA to use as a molecular weight marker. Before you can do the digests, you'll need to  perform a DNA assay and do some calculations. In the next lab period, you'll run your cut DNA on a gel.

Background

The virtual digest page gives the background for plasmid restriction digests; you should read that page and complete the assignment before you do the digests described here.

The page you're on now is mainly about how to do the calculations and the technique for performing restriction digests.

Methods

DNA assay

It's best to know your starting DNA concentration so you can get the restriction digest and gel right, so we often use a fluorescent DNA assay to measure the concentration of purified plasmid DNA. However, the plasmid purification results in Bio 6B are generally consistent, so we'll skip the assay this time. Let's asume that you will have just enough plasmid DNA to be visible on the gel, so you will want to maximize the amount of DNA that you get on the gel. The maximum volume that you can fit in one well of your DNA gel is about 25 μl, so that will be the volume of each restriction digest.

Calculations for a plasmid restriction digest

You'll do 8 restriction digests, as shown in the table below (we're doing 8 tubes today because the gel you run next time will have 8 wells). To cut your DNA, you need to set up each reaction tube with the appropriate amounts following ingredients:

DNA
This is what gets cut. You'll have 5 samples of DNA for this experiment: pARO180, 3 versions of pGLO, and Lambda (λ) bacteriophage. You’ll use electrophoresis to look at the DNA after the reaction is done, so for this experiment the correct amount of DNA to cut is the amount (in micrograms or nanograms) that will look good on the gel. If there's not enough DNA, you won't see all the bands; if there's too much, the bands will be smeared and you won't be able to determine their size. In this experiment, the concentration of the plasmid samples will be unknown, but the lambda DNA will be known.
Restriction enzymes
In Bio 6B, we use various restriction enzymes to cut the DNA, including as SspI, PvuI, EcoRI and HindIII. If you know the mass of DNA you are trying to cut in micrograms, you can calculate the amount of enzyme to use. Enzyme concentrations are given in terms of units per microliter, where one unit of enzyme is defined as enough to cut a certain amount of DNA.
Enzyme buffer
Every enzyme needs the appropriate buffer. Enzymes are proteins, and they depend on having the right 3-dimensional shape to do their job. Since the shape of a protein is controlled in part by weak interactions such as hydrogen bonds, it can be altered by changes in pH or salt concentration. When you buy an enzyme, it comes with a buffer that ensures the right environment for the enzyme. Since you're combining DNA, buffer, water, and enzyme in a tube, each ingredient dilutes the others. The buffer usually comes as a 10x concentrate, and by the time you put all the ingredients in the tube, the final buffer concentration should be 1x. The buffers for today's lab are: SspI uses Tango buffer; PvuI uses Anza buffer; Hind III uses 2.1 buffer. Each enzyme would probably work with the other buffers, but I haven't tested this.
Water
You’ll generally need to add a little water to make the concentrations of everything else come out right.

To figure out how much of each ingredient to put in the tube, you’ll have to do a little math. Look at the calculations page for some tips. For the plasmid DNA samples, Do all your calculations and show them to the instructor before you’re ready to start doing the lab work. Once you have all your results, put them in a table like this:

 Tube# 1  2  3  4  5  6  7 8
 DNA pARO180 pARO180 pGLO Green  pGLO Green pGLO Blue pGLO Non-F pGLO non-F λ (Lambda)
ng DNA unknown unknown unknown unknown unknown unknown unknown 500 ng
 Enzyme PvuI SspI PvuI SspI SspI PvuI SspI Hind III
 Water  0 μl   0 μl  0 μl  0 μl   0 μl  0 μl   0 μl  
 pARO180 DNA      0 μl 0 μl  0 μl 0 μl  0 μl 0 μl
pGLO Green 0 μl 0 μl     0 μl 0 μl 0 μl 0 μl
 pGLO Blue  0 μl 0 μl   0 μl  0 μl    0 μl  0 μl  0 μl
pGLO Non-F 0 μl 0 μl 0 μl 0 μl 0 μl      0 μl 
λ DNA 0 μl 0 μl 0 μl 0 μl 0 μl 0 μl 0 μl  
Anza Buffer   0 μl    0 μl  0 μl   0 μl 0 μl
Tango Buffer 0 μl   0 μl     0 μl   0 μl 
2.1 Buffer 0 μl 0 μl 0 μl 0 μl 0 μl 0 μl 0 μl  
SspI 0 μl  0.4 μl  0 μl  0.4 μl 0.4 μl 0 μl  0.4 μl  0 μl 
PvuI 0.4 μl 0 μl  0.4 μl 0 μl   0 μl  0.4 μl  0 μl 0 μl
Hind III 0 μl 0 μl 0 μl 0 μl 0 μl 0 μl 0 μl  
Total volume 25 μl 25 μl 25 μl 25 μl 25 μl 25 μl 25 μl 25 μl

The digests shown in the table should give you clear differences among the plasmids when you run the digested DNA on a gel.

To figure out the numbers for the table, work through the calculations below.

Reaction volume

Since you're planning to run your restriction digest on a gel, the maximum useful volume is the maximum that will easily fit in one well of our DNA gels: about 25 μl. You could use a smaller volume (if your DNA is concentrated enough), but if it's too small it becomes more difficult to pipet everything. For this lab, use 25 μl for each reaction.

How much plasmid DNA?

You need enough to be clearly visible on the gel, but not so much that it will overload the gel and make a big smear. Since the concentrations of the plasmids will be unknown, you should maximize the amount of DNA. Simply use 0.4 μl of enzyme, the appropriate amount of buffer, and make up the rest of the 25 μl reaction volume with your DNA solution. You don't need to add water unless you want to dilute your DNA.

How much λ DNA?

The starting concentration of the lambda DNA is 250 ng/μl. Calculate the number of microliters to give you 500 ng DNA for the digest.

If there's going to be room on the gel, you should do a lane of uncut λ DNA (the gel will have 8 lanes).The purpose of the uncut λ tube is to serve as a negative control so you will be able to compare your cut to the uncut DNA. In principle, you could do that by simply loading some of the concentrated stock solution on the gel, but the high concentration would mean that you would end up loading a very small volume on the gel to get the desired 250 ng. Loading less than 5 μl in a well on the DNA gel doesn't work very well, because it's not enough volume to spread out in the well and form clear bands. This probably won't be an issue with your plasmid DNA samples, because the concentration is likely to be lower.

How much enzyme?

You need enough Units of enzyme to cut the mass of DNA in your reaction tube. Enzyme activity is defined in terms of arbitrarily chosen units for each enzyme. For these restriction enzymes, the unit is defined as: 1 Unit will completely cut 1.0 μg of lambda DNA in 1 hour at 37° C.

HindIII stock solution: 20,000 Units/ml.

Take a look at the calculations page, and write out your equation for how many μl of enzyme you need for each reaction:

Minimum enzyme amount: Need at least ____________μl enzyme. (This is a very small amount.)

In practice, it’s a good idea to use some extra restriction enzyme, to make sure all the DNA gets cut. For this reaction (and other restriction digests in Biio 6B), multiply the minimum enzyme amount (which you just calculated) by 5. So you’ll add ____________μl HindIII. Adding extra restriction enzyme doesn't do any harm, because the enzyme is very specific about cutting DNA only at its specific restriction sites.

For the the plasmids, the mass of DNA will be unknown, so you can't calculate the enzyme amount. It's going to be a tiny amount, so I just chose 0.4 μl of enzyme, which is the smallest amount I think you can comfortably pipet.

How much buffer?

Each enzyme comes with a specific buffer. Make sure you get the correct one for each each enzyme. The buffer comes as a 10x concentrate, and you need it to be diluted to 1x final concentration in your reaction tube. To figure out the amount, use C1V1=C2V2. In this case,

  • C1= initial concentration = 10x.
  • V1= initial volume = this is what you’re trying to solve for; the answer should be in μl.
  • C2= final concentration = 1x.
  • V2= final volume of the restriction digest. The final volume is what’s in the tube after you’ve added everything, including water.

Calculate the volume of buffer for each reaction. Since all your reaction volumes are the same, the buffer volume is the same.

How much water?

You’ve already figured out how much enzyme, DNA, and buffer for the lambda digest. You know the final volume. After you’ve figured all this out, calculate how much water you’ll need to add to bring the volume of each reaction up to the appropriate amount. For the plasmid digests, don't add any water, because you don't want to dilute your DNA.

Be ready to do calculations like these on a quiz. You should know that you always need a buffer for the enzyme, and sometimes you aren’t given the final volume.

Check your numbers with the instructor.

Set up the reactions

Materials:

With the calculations out of the way, the rest is easy. For each table, obtain the following:

  • Pipetmen, yellow tips, and a beaker for waste tips
  • Rack for microfuge tubes
  • Small tub or cup filled with ice
  • One 500 μl microfuge tube for each reaction (the tiny tubes, not the larger 1.5 ml size)
  • Sterile water (comes in a glass bottle)
  • Lambda DNA
  • Your purified plasmid samples
  • 10x enzyme buffer (You can use the same buffer for all the enzymes, but there are several different buffers available. Write down which buffer you are using: e.g., NEBuffer 2.1 or Anza buffer.)

The instructor will give you the enzymes when you’re ready -- after you’ve pipetted everything else into the tubes:

  • Ssp I restriction enzyme
  • Hind III restriction enzyme
  • Pvu I restriction enzyme

We will only have one or two tubes of each enzyme, so you'll have to take turns.

Set up the reactions

It’s essential to follow some simple rules as you set up the reactions:

  • Keep everything cold. Keep all the ingredients (except the water) and the assembled reaction tubes on ice until it’s completely assembled and you’re ready to incubate it.
  • Keep the enzymes on ice. The enzymes should stay in their dedicated ice buckets throughout the lab. Only take the enzyme tubes off the ice for the time it takes you to pipet the enzyme. This shouldn’t take you more than 10 seconds.
  • Add the ingredients in the order listed in the table above. Add the enzyme last. Start with the water, because it’s a relatively large volume; it’s difficult to pipet a small volume into an empty tube. The enzyme could be destroyed if you add it before you add the buffer, so it must go in last.
  • Never use a pipet tip twice — change tips every time. This prevents cross-contamination.
  • Keep all the ingredients in the bottom of their microfuge tubes, and mix each one with the pipet tip as you take it out. You need to see the tiny drop of liquid come out of the tip and mix with the ingredients already in the tube. You may need to use the centrifuge to get the solution to the bottom of the tube.
  • Once you’ve assembled your materials, go ahead and set up your reaction according to the amounts you calculated. All you need to do is pipet 4 or 5 ingredients into each tube, and you’re done! As mentioned earlier, the hard thing is figuring out what goes into the tube.
  • After you pipet all the ingredients, incubate your reaction in the PCR machine at 37° C. We’ll program the machine to run for one hour at 37° C, and then heat up to 80° C for 3 minutes to destroy (heat-kill) the enzyme. The program on the PCR machine is called Cut & Kill.

Before you leave

  • You should have 8 tubes of DNA from your restriction digests in the freezer or in the thermal cycler, plus any leftover uncut DNA. You need all these tubes! You’ll use them when you run a gel in the next lab period.
  • You may have tubes with very small amounts of leftover DNA or buffers; check with instructor before throwing any of it away.
  • Waste micro tubes and pipet tips go in the biohazard trash.
  • Dirty beakers (used for collecting waste pipet tips) go into a tub for dirty glassware. Dump the tips out first!
  • Dump your ice down the sink, and put everything else back where you got it.

Review

Terms and concepts

  • Linearize
  • Molecular weight marker
  • Restriction enzyme
  • Restriction site
  • Supercoiled vs. linear DNA

Review questions

  1. Why does it matter if the DNA on a gel is supercoiled?
  2. Why is it important to add the enzyme last when setting up a restriction digest?
  3. On a quiz, be ready to calculate the amounts of all the ingredients in a restriction digest, as shown on this page.

References

Bio 6B Flickr site. Find your gel photo and compare it to the photos of other groups.

Restriction Enzymes from PDB-101. Brief description, with good images. This is an excellent site.

DNA Restriction from DNA Learning Center. Brief animated description of restriction, sticky ends, and ligation.

How to Identify Supercoils, Nicks and Circles in Plasmid Preps BiteSize Bio

 

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